Constrained selected reaction monitoring: Quantification of selected post-translational modifications and protein isoforms

Xiaoqian Liu, Zhicheng Jin, Richard O'Brien, Joan Bathon, Harry C. Dietz, Eric Grote, Jennifer E. Van Eyk

Research output: Contribution to journalArticlepeer-review

Abstract

Selected reaction monitoring (SRM) is a mass spectrometry method that can target signature peptides to provide for the detection and quantitation of specific proteins in complex biological samples. When quantifying a protein, multiple peptides are generated using a specific protease such as trypsin, thereby allowing a choice of signature peptides with robust signals. In contrast, signature peptide selection can be constrained when the goal is to monitor a specific post-translational modification (PTM) or protein isoform, as the signature peptide must include the amino acid residue(s) of PTM attachment or sequence variation. This can force the selection of a signature peptide with a weak SRM response or one that is confounded by high background. In this article, we discuss steps that can be optimized to maximize peptide selection and assay performance of constrained SRM assays, including tuning instrument parameters, fragmenting product ions, using a different protease, and enriching the sample. Examples are provided for phosphorylated or citrullinated peptides and protein isoforms.

Original languageEnglish (US)
Pages (from-to)304-312
Number of pages9
JournalMethods
Volume61
Issue number3
DOIs
StatePublished - Jun 15 2013
Externally publishedYes

Keywords

  • Post-translational modification
  • Protein isoform
  • Quantification
  • Selected reaction monitoring

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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